|Ahead of print publication
Combined effect of oral famotidine and cimetidine on the survival of lethally irradiated mice: An in vivo study
Karim Afsar dizaj1, Ali Shabestani Monfared2, Hossein Mozdarani3, Ali Naeiji4, Abolfazl Razzaghdoust5, Karimollah Hajian-tilaki6, Bahareh Aboufazeli1, Fatemeh Niksirat7, Sajad Borzoueisileh8
1 Student Research Committee, Babol University of Medical Sciences, Babol, Iran
2 Cancer Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
3 Department of Medical Genetics, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
4 Radiology Technology Department, Faculty of Paramedical Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5 Urology and Nephrology Research Center, Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran
6 Department of Statistic and Epidmiology, School of Medicine, Babol University of Medical Sciences, Babol, Iran
7 Cancer Research Center; Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
8 Student Research Committee; Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
|Date of Submission||19-May-2019|
|Date of Decision||10-Aug-2019|
|Date of Acceptance||26-Nov-2019|
|Date of Web Publication||03-Nov-2020|
Ali Shabestani Monfared,
Babol University of Medical Sciences, Ganjafrooz Street, Babol
Source of Support: None, Conflict of Interest: None
Aims: The study aims at evaluating the effects of the combinatory famotidine/cimetidine diet on radiated mice's survival.
Materials and Methods: Two hundred and seventy male mice were categorized into 11 groups, a number of which were comprised of subgroups too. The groups under analysis were posed to varying doses of gamma-radiation, including 6, 7, 8, and 9 Gy, followed by treatments using various drug doses 2, 4, and 8 mg/kg, with survival fractions as long as a month after irradiation being measured and recorded.
Results: LD50/30 was calculated as 7.47 Gy for the group with radiation only. Following mouse treatment with a concentration of 4 and 20 mg/kg for famotidine and cimetidine, respectively, the survival fraction for the mice grew significantly compared to LD50/30. The combinatory famotidine/cimetidine diet had a higher dose-reduction factor (DRF) than single doses of the drug in radioprotection. The DRF for combinatory famotidine/cimetidine, famotidine, and cimetidine diets was 08.09, 1.1, and 1.01, respectively.
Conclusions: Results imply that the combined regimen of famotidine + cimetidine in radioprotection had no significant higher DRF than with regimens including each of them separately. In addition, we did not find a synergic effect of combined oral famotidine and cimetidine on irradiated mice.
Keywords: Cimetidine, famotidine, radiation, survival fraction
|How to cite this URL:|
dizaj KA, Monfared AS, Mozdarani H, Naeiji A, Razzaghdoust A, Hajian-tilaki K, Aboufazeli B, Niksirat F, Borzoueisileh S. Combined effect of oral famotidine and cimetidine on the survival of lethally irradiated mice: An in vivo study. J Can Res Ther [Epub ahead of print] [cited 2020 Dec 3]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=299885
| > Introduction|| |
Ionizing radiations can throw out electrons from the atom  which, in living organisms, could cause cell damages. Cells could repair most of these damages, but unrepaired damages could result in cell death or mutation. Acute extensive damages in multicellular organisms, induced by radiation, could lead to organ or tissue damage or resulted in death. The response of different individuals against ionizing radiation could be different. Several reasons for this difference have already been introduced.
We could manipulate the damages of radiation by radioprotectors and radiosensitizers. Many agents were studied in this way, and amifostine has a Food and Drug Administration approval for use in some of the radiotherapy patients for normal cell protection.
Cimetidine and famotidine belong to antagonists to the histamine H2 receptor whose efficacy in protection against radiation was studied. Furthermore, these drugs scavenge the free radicals which help the cells to modulate the indirect effects of ionizing radiation., Furthermore, cimetidine is believed to possess immunomodulatory properties.
Mozdarani and Gharbali reported a reduction in radiation-induced micronuclei (MNs) by cimetidine. Zeng et al. have suggested an increase in cell-mediated immunity by cimetidine in combination with radiation for the treatment of nasopharyngeal cancer. Nagano et al. reported two cases of metastatic renal cell carcinoma which were successfully treated with cimetidine. In 1998, the protective effect of cimetidine against benzene and fast MNs, induced by neutron, in the cells of mouse bone marrow , was reported, and in 2002, Kojima et al. studied protective effects of cimetidine on MNs and apoptosis in human peripheral blood lymphocytes. In 2015, decreases in radiation-induced damages of the thyroid gland by cimetidine were reported in mice.
Famotidine is another antagonist of the histamine H2 receptor whose radioprotective effects were studied. In 2014, Razzaghdoust et al. reported that an increase in radiotherapy efficacy and survival times by famotidine might be achieved. Furthermore, famotidine were used for rectal mucosa protection in patients suffering from prostate cancer who were treated with radiotherapy. The combined effect of famotidine and Vitamin C was reported against MNs exposed to radiation in erythrocytes of mouse bone marrow.
Cimetidine and famotidine belong to antagonists to the histamine H2 receptor whose efficacy in protection against radiation was studied, whereas oral administration effectiveness for improving the survival of lethally irradiated mice has not been studied yet. The last endpoint in our study was survival of animals which measures the direct outcome of radiation exposure. Furthermore, it is clinically more important than MNs which were considered in other studies. One of the most important specifications of a radioprotector is its capability for oral use. Furthermore, the combination of famotidine and cimetidine has not been evaluated so far. Because using combined agent regimens is a favorable methodology to maximize radioprotection and still keep the negative effects at a nominal rate, in this study, the combined effects of famotidine and cimetidine on irradiated mouse survival were studied.
| > Materials and Methods|| |
Animal care and handling
A total of 270 male NMRI mice, 6–8 weeks old and with 28 ± 3 g of weight, were provided from the animal laboratory of Babol University of Medical Sciences. The animals were quarantined in the metal cage for at least 1 week before the experiment. The animals were fed with standard mouse pellet and water ad libitum during the 30 days' study period and were kept in a controlled environment with a temperature of 22°C ± 1°C, with 50%–60% humidity and 12-h light/dark cycles.
All the ethical points of the current study have been considered and accepted by the Committee of Ethics at Babol University of Medical Sciences, Babol, Iran and the experiments were conducted in accordance with the regulations of the Care and Use of Laboratory Animals.
Famotidine and cimetidine powders were purchased from QQ. Doses of 2, 4, and 8 mg/kg famotidine, 10, 20, and 40 mg/kg cimetidine, and the combination of both (ratio: 1:5; famotidine/cimetidine) were dissolved in distilled water based on the results of a pervious study by Mozdarani et al. Then, aliquots of different concentrations were orally given to animals with a gavage needle every 12 h for 3 days, and 2–3 h before irradiation, as well.
The mice in different irradiated groups were restrained in a metal cage and were exposed to whole-body irradiation with the doses of 6, 7, 8, 9 Gy and LD50 in a single fraction using a Cobalt-60 machine (Theratron II, 780 C, Canada). The dose rate for the experiment was 91cGy/min, and the dosage was calculated at an 80-cm source-to-skin distance. The animals were returned to the home cage after irradiation, and their mortality were measured in the following 30 days.
In this study, 270 animals were randomly divided as follows: ten animals in each subgroup, based on their experimental treatment:
- Group 1: Sham control group: Mice in this group were neither treated nor irradiated
- Group 2: Irradiated mice with various doses (6, 7, 8, and 9 Gy): Mice in this group were exposed to whole-body gamma-radiation with a single dose
- Group 3: Famotidine control, receiving the highest dose chosen (8 mg/kg)
- Group 4: Cimetidine control with the highest selected dose (40 mg/kg)
- Group 5: Radiation (7.47 Gy) + varying dosages of famotidine (2, 4, and 8 mg/kg)
- Group 6: Radiation (7.47 Gy) + varying dosages of cimetidine (10, 20, and 40 mg/kg)
- Group 7: Famotidine (4 mg/kg) + varying dosages of radiation (6, 7, 8, and 9 Gy)
- Group 8: Cimetidine (20 mg/kg) + varying dosages of radiation (6, 7, 8, and 9 Gy)
- Group 9: Irradiated nontreated mice (7.47 Gy) (LD50/30 group)
- Group 10: Control group for the combination regimen of famotidine and cimetidine (1–5 for famotidine-to-cimetidine ratio)
- Group 11: Combination regimen of famotidine and cimetidine + varying dosages of radiation (6, 7, 8, and 9 Gy).
Determination of LD50/30 for irradiation
Before investigating the protective effect of drugs, it was necessary to measure LD50/30. Four groups of ten mice were induced using four different doses of radiation (6, 7, 8, and 9 Gy). Following irradiation, the animals were screened for 30 days, and their daily mortality was evaluated during this period.
Determination of the optimal radioprotective dose of drugs by the use of survival assay
To determine the optimal dose, the lowest dose of drugs which significantly increases the survival rate compared to the non-treated group should be used. In our study, various doses of famotidine (2, 4, and 8 mg/kg) and cimetidine (10, 20, and 40 mg/kg) were gavaged to the mice every 12 h for 3 days, and 2–3 h before irradiation, as well. Various doses of famotidine (2, 4, and 8 mg/kg) and cimetidine (10, 20, and 40 mg/kg) were gavaged to the mice every 12 h for 3 days, and 2–3 h before irradiation, as well. Then, the mice were exposed to a dose of gamma-rays equal to the LD50/30. An irradiated nontreated group and two treated groups with a maximum dose of famotidine or cimetidine were considered as controls. The survival fraction was calculated according to the daily record of the mortality during 30 days. In the end, the obtained optimum dose of each drug was selected to analyze dose-reduction factor (DRF) and using in the combination regimen.
Determination of dose-reduction factor
The DRF is a ratio which can be considered as the radioprotective capability of an agent. The optimal radioprotective dose of famotidine and cimetidine alone and in combination with each other was orally administered to measure the DRF. Then, the mice were exposed to the different single doses of whole-body gamma-irradiation (6, 7, 8, and 9 Gy). LD50/30 was determined in different groups by measuring the rate of mortality during 30 days. Furthermore, DRF was calculated by dividing the LD50/30 of famotidine or cimetidine, as well as their combination in treated mice by LD50/30 of irradiated nontreated mice.
The Probit statistical analysis test was used to determine both LD50/30 and DRF. The statistical significance of the differences in survival rates compared to controls was investigated according to the log-rank test. Moreover, the significance of any intergroup differences in the survival rate was investigated by log-rank test. Furthermore, data on 30-day survival or mortality of animals were analyzed using the survival curves of Kaplan–Meier. In all instances, P values lower than 0.05 were deemed significant.
| > Results|| |
LD50 is 7.47 Gy
Based on Probit Analysis, the LD50 was 7.47 Gy without oral famotidine and cimetidine administrations. The data are shown in [Figure 1].
|Figure 1: The survival fraction for different doses of radiation. The P values compared to 0 Gy group by log-rank test|
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Survival fraction of different groups
The optimal dosage of famotidine and cimetidine was determined by administration of their different doses alongside with LD50/30 radiation, which was compared to LD50/30 without drug treatment. The optimal doses of famotidine and cimetidine were determined as 4 and 20 mg/kg, respectively. The data are shown in [Figure 2].
|Figure 2: The survival fraction of different treatment doses of famotidine and cimetidine along with LD50 irradiation (7.47 Gy). P values compared to LD50 group by log-rank test|
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Dose-reduction factor calculations
The measurements of survival fraction of the animal in the presence of famotidine, cimetidine, and combined regimen of famotidine + cimetidine are shown in [Figure 3].
|Figure 3: The survival fraction of (a) famotidine (Fam), (b) cimetidine (Cim), and (c) combined famotidine + cimetidine (Fam + Cim) in different radiation doses for LD50 and dose-reduction factor achievements. The famotidine and cimetidine doses were 4 and 20 mg/kg of mouse weight, respectively. The P values are compared to control group by log-rank test|
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The LD50/30 of radiation, radiation + famotidine, radiation + cimetidine, and radiation + famotidine + cimetidine measured by Probit Analysis is shown in [Table 1]. By dividing the LD50 of mice in the presence of drugs to radiation alone, the DRF is measured and presented in [Table 1].
|Table 1: LD50 and dose-reduction factor for radiation, radiation + famotidine, radiation + cimetidine, and radiation + famotidine+cimetidine|
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| > Discussion|| |
In this in vivo study, the combined and separate effects of famotidine and cimetidine on the survival of irradiated mice were assessed for the first time. The survival fraction, LD50/30, and DRF of different doses of these drugs and different doses of radiation were compared to each other and sham control group.
Radiation could ionize the molecules of living organisms, resulting in several consequences. Some situations could modulate these responses against ionizing radiation. One of these modulating factors is radioprotectors. To compare the strength of different radioprotectors, DRF is a useful variable. The DRF calculation needs a common endpoint which was LD50/30 in our study. The LD50/30 of radiation alone in our study population was determined as 7.47 Gy. Jeena et al. reported an LD50/30 of 7.12 Gy for their study population  and Mozdarani et al. reported 7.23 Gy  which are somewhat in agreement, while their difference could be as the result of different situations such as dose rate of radiations.
Our results showed that the low concentration of famotidine and cimetidine could not protect the mice significantly against irradiation. However, as the concentration of these drugs increased, the difference in survival fraction significantly increased. 2 mg/kg famotidine and 10 mg/kg cimetidine enhanced the survival fraction, though it was not statistically significant. As the concentration of famotidine and cimetidine increased to 4 and 20 mg/kg, respectively, the survival fraction for animals grew significantly higher than that of the LD50/30 group. The further increase in concentration to 8 and 40 mg/kg for famotidine and cimetidine, respectively, resulted in 100% survival, despite receiving an LD50 radiation dose. The 8 and 40 mg/kg concentration for famotidine and cimetidine, respectively, without irradiation, did not affect the survival of animals, indicating that these concentrations are not lethal for mice by oral administration. Mozdarani et al. reported that the 15 mg/kg concentration of cimetidine could slightly better modulate the radiation-induced MNs compared to 7.5 and 30 mg/kg. Their optimal radioprotector concentration is different from our study mainly because of the different radiation types and endpoint and studied cells. They have used neutron while we used gamma-rays both of which are important. Furthermore, their final endpoint was MNs, but our endpoint was a 50% survival of animals which clinically is more important than Mozdarani et al. study. In another study, the 30 mg/kg cimetidine concentration was more effective than 10 mg/kg against genotoxic effects of MNs induced by benzene in the erythrocytes of mouse bone marrow.
As [Figure 3] shown after treating mice with famotidine, the survival fraction was significantly different to controls after receiving 9 Gy. However, the survival fraction of the cimetidine group was significantly different to controls at 8 Gy irradiations. This means that lower doses of radiation could decrease the survival of mice in the presence of cimetidine than famotidine, implying that the famotidine at these concentrations and these dose ranges protects mice more than the cimetidine against ionizing radiation. In a study in 2003, the author measured the DRF of three H2 antagonists and concluded that famotidine is a better radioprotector than ranitidine and cimetidine. Their final endpoint was MN, but our endpoint was LD50 which clinically is more important, but the MN count is not directly a clinically important factor.
Comparison of DRF for radiation alone, radiation + famotidine, radiation + cimetidine, and radiation + famotidine + cimetidine implies that the combined regimen of famotidine + cimetidine in radioprotection had a higher DRF than each of them separately. The DRF of combined famotidine + cimetidine, famotidine, and cimetidine groups was 1.09, 1.08, and 1.01, respectively, which is rather lower than the study of Mozdarani in 2003. This difference in the results of our study with Mozdarani et al. could be due to the different in study designs and endpoints, where we studied the survival fraction they assessed MNs in 1000 cells. The LD50/30 of these groups was 8.16, 8.1, and 7.57, respectively. Low values of DRF reported in our study, not showing high radioprotective effects, might be due to a different endpoint, drug dose selection, and inappropriate drug delivery, for which we suggest considering these variables for the design of upcoming studies.
| > Conclusion|| |
Famotidine and cimetidine individually and in combination with each other provide radiation protection. Combined regimen of these drugs in radioprotection had no significant higher DRF than with regimens including each of them separately. The efficiency of these two drugs in scavenging free radicals may indicate that famotidine and cimetidine are hydroxyl radical scavengers.
Financial support and sponsorship
This work is financially supported by the Vice-Chancellor for Research and Technology, Babol University of Medical Sciences, Babol, Iran, under Grant number 2321.
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Brewer S. Irradiation effects on meat color – A review. Meat Sci 2004;68:1-7.
Shabestani Monfared A, Borzoueisileh S, Zabihi E, Amiri M, Abedian S. Predicting factors of radiosensitivity in individual radiotherapy. J Babol Univ Med Sci 2015;17:67-73.
Grdina DJ, Murley JS, Kataoka Y. Radioprotectants: Current status and new directions. Oncology 2002;63 Suppl 2:2-10.
Ching TL, Haenen GR, Bast A. Cimetidine and other H2 receptor antagonists as powerful hydroxyl radical scavengers. Chem Biol Interact 1993;86:119-27.
Mortazavi S, Mozdarani H. Deep space missions and the issue of overcoming the problem of space radiation. Int J Radiat Res 2013;11:199-202.
Sinha S, Relhan V, Garg VK. Immunomodulators in warts: Unexplored or ineffective? Indian J Dermatol 2015;60:118-29.
] [Full text]
Mozdarani H, Gharbali A. Radioprotective effects of cimetidine in mouse bone marrow cells exposed to γ-rays as assayed by the micronucleus test. Int J Radiat Biol 1993;64:189-94.
Zeng P, Xiao J, Lei Y. Cell-mediated immune function in NPC patients treated with cimetidine. Zhonghua Zhong Liu Za Zhi 1995;17:223-5.
Nagano T, Matsuda H, Park YC, Kurita T. Successful treatment of metastatic renal cell carcinoma with cimetidine--report of two cases. Nihon Hinyokika Gakkai Zasshi 1996;87:1201-4.
Mozdarani H, Kamali S. Antigenotoxic effects of cimetidine against benzene induced micronuclei in mouse bone marrow erythrocytes. Toxicol Lett 1998;99:53-61.
Mozdarani H, Khoshbin-Khoshnazar AR.In vivo
protection by cimetidine against fast neutron-induced micronuclei in mouse bone marrow cells. Cancer Lett 1998;124:65-71.
Kojima Y, Kondo T, Zhao QL, Shoji M, Futatsuya R. Protective effects of cimetidine on radiation-induced micronuclei and apoptosis in human peripheral blood lymphocytes. Free Radic Res 2002;36:255-63.
Fazelipour S, Kiaei SB, Tootian Z, Haeri SA, Zehtabvar O, Sadeghinezhad J. Cimetidine administration decreases radiogenic damage on the thyroid gland in mice. Int J Radiat Biol 2015;91:218-23.
Razzaghdoust A, Mozdarani H, Mofid B, Aghamiri SM, Heidari AH. Reduction in radiation-induced lymphocytopenia by famotidine in patients undergoing radiotherapy for prostate cancer. Prostate 2014;74:41-7.
Razzaghdoust A, Mozdarani H, Mofid B. Famotidine as a radioprotector for rectal mucosa in prostate cancer patients treated with radiotherapy: Phase I/II randomized placebo-controlled trial. Strahlenther Onkol 2014;190:739-44.
Zangeneh M, Mozdarani H, Mahmoudzadeh A. Potent radioprotective effects of combined regimens of famotidine and Vitamin C against radiation-induced micronuclei in mouse bone marrow erythrocytes. Radiat Environ Biophys 2015;54:175-81.
Mozdarani H, Salimi M, Froughizadeh M. Effect of cimetidine and famotidine on survival of lethally gamma irradiated mice. Int J Radiat Res 2008;5:187-94.
Jeena K, Liju VB, Ramanath V, Kuttan R. Protection against whole body γ-irradiation induced oxidative stress and clastogenic damage in mice by ginger essential oil. Asian Pac J Cancer Prev 2016;17:1325-32.
Mozdarani H. Radioprotective properties of histamine H2 receptor antagonists: Present and future prospects. J Radiat Res 2003;44:145-9.
[Figure 1], [Figure 2], [Figure 3]